Method of stabilizing protein

ABSTRACT

The present inventors revealed that deamidation of an antibody can be suppressed without influencing its activity by substituting a glycine that is located adjacent to an asparagine with another amino acid.

TECHNICAL FIELD

The present invention relates to a method for improving proteinstability. Specifically, the present invention relates to a method forstabilizing proteins comprising the step of substituting the amino acidthat is located adjacent to the amino acid being deamidated in a proteinwith another amino acid.

BACKGROUND ART

Gradual deamidation of amino acids, such as asparagine, in proteins overtime is mentioned as a cause of the reduction in protein stability. Whenproteins, particularly antibodies, are used as pharmaceutical agents forvarious diseases, they are required to be stable over a long period.However, the activity of antibody decreases with time. The cause forreduction in activity varies in antibodies, and deamidation of aminoacids, such as asparagine, comprised in the antibody is also mentionedas one of the causes.

Therefore, proteins can be stabilized by suppressing deamidation ofasparagines. Thus, research on suppressing deamidation of asparagine hasbeen conducted. The substitution of asparagine with another amino acidby site-directed mutagenesis is considered the most certain method toprevent deamidation of proteins. However, this substitution has thepotential to influence protein activity. For example, when theasparagine is located in the complementary determining region (CDR) ofan antibody, such substitution is reported to affect the antibodybinding affinity (Presta L. et al., Thromb. Haemost. 85: 379-389, 2001).An anti-human tissue factor (TF) antibody that is expected to inhibitthrombus formation without inhibiting the extrinsic blood coagulationreaction via the inhibition of Factor X activation mediated by TF in theintrinsic blood coagulation reaction is known in the art (WO 99/51743).However, this antibody has not been formulated as a pharmaceuticalpreparation and its activity reduces over time under antibodydestabilizing conditions. The deamidation of anti-human TF antibody issupposed to be a factor of such reduction.

Thus, a method to suppress deamidation of asparagine without influencingantibody activity has been desired in the art.

DISCLOSURE OF THE INVENTION

Reduction in protein activity is a very important problem from themedical and pharmaceutical perspectives. Particularly, antibodies thatare stable for a long time and which can be used as pharmaceuticalagents are clinically desired. To stabilize antibodies, it isparticularly required to suppress deamidation over time of amino acidssuch as asparagine, mainly, those readily deamidated in Asn-Glysequences.

Conventionally, methods to suppress deamidation by altering amino acidsin proteins is a useful technique to improve the value, quality and suchof pharmaceuticals. Such methods increase the option in the formulationof pharmaceutical preparations, and thus facilitate application of theproteins to various drug forms and administration routes. Therefore, thepurpose of the present invention is to provide a method to suppressdeamidation of asparagine without influencing the activity of proteins,particularly antibodies.

The present inventors diligently conducted research focusing onanti-human TF antibody, which use as a pharmaceutical is expected in theart. The antibody was used as an example of a protein for developing amethod to suppress deamidation of asparagine without affecting theprotein activity. First, a mutated anti-human TF antibody was expressedas a recombinant wherein asparagine that may be deamidated yet existingin the CDR, is substituted with aspartic acid. The TF binding activityof anti-human TF antibody was suggested to decrease significantly due tothe deamidation of Asn54 existing in the CDR2 region of the anti-humanTF antibody heavy chain (H chain). The amino acid adjacent to Asn54 inthe CDR2 region of anti-human TF antibody heavy chain is Gly55. Thesetwo amino acids form a primary sequence Asn-Gly that is easilydeamidated. Therefore, the possibility to suppress deamidation of Asn54by substituting this Gly55 with another amino acid was considered. Thus,the present inventors prepared mutants wherein the glycine adjacent tothe asparagine was substituted with other amino acids to measure theirbinding activities. As a result, it was discovered that the substitutionof glycine that is located adjacent to asparagine with other amino acidsdid not reduce the activity, and also suppressed the known instabilitydue to deamidation.

Thus, the present inventors found that antibody activity is uninfluencedby the substitution of glycine that is located adjacent to asparaginewith other amino acids, instead of the substitution of the asparagineitself, and thereby completed the present invention.

Specifically, the present invention provides the following:

-   (1) a method for stabilizing a protein, which comprises the step of    substituting an amino acid that is located adjacent to an amino acid    being deamidated with another amino acid;-   (2) the method for stabilizing a protein of (1), wherein the amino    acid being deamidated is asparagine;-   (3) the method for stabilizing a protein of (1), wherein the amino    acid that is located adjacent to the C-terminal side of the amino    acid being deamidated is glycine;-   (4) the method for stabilizing a protein of any one of (1) to (3),    wherein the protein is an antibody;-   (5) the method for stabilizing a protein of (4), wherein the    antibody is humanized antibody;-   (6) the method for stabilizing a protein of (4) or (5), wherein the    amino acid being deamidated exists in the complementary determining    region (CDR);-   (7) the method for stabilizing a protein of (6), wherein the    complementary determining region (CDR) is CDR2;-   (8) the method for stabilizing a protein of any one of (1) to (3),    wherein the protein is an antigen binding protein;-   (9) the method for stabilizing a protein of any one of (1) to (3),    wherein the protein belongs to the immunoglobulin superfamily;-   (10) the method for stabilizing a protein of any one of (1) to (3),    wherein the protein is a pharmaceutical agent;-   (11) a protein stabilized by the method of any one of (1) to (10);    and-   (12) the stabilized protein of (11) whose antigen binding activity    is 70% or more of the activity before the amino acid substitution.

The terms described in the specification are defined as follows.However, it should be understood that the definitions are provided tofacilitate understanding of the terms used herein and are not to beconstrued as limiting the present invention.

The term “protein” herein refers to recombinant proteins, naturalproteins and synthetic peptides prepared by artificially combining aminoacids, which proteins and peptides consist of five amino acids or more.Proteins consist of amino acid sequences having preferably 14 residuesor more, more preferably 30 residues or more, and much more preferably50 residues or more.

The term “antibody” used in the stabilization method of the presentinvention is used in the broadest sense, and includes monoclonalantibodies (including full-length monoclonal antibodies), polyclonalantibodies, mutant antibodies, antibody fragments (for example, Fab,F(ab′)₂ and Fv) and multispecific antibodies (for example, bispecificantibodies) as long as they have the desired biological activity.Antibodies (Ab) and immunoglobulins (Ig) are glycoproteins that sharethe same structural features. Antibodies show a specific binding abilityto a certain antigen, while immunoglobulins include antibodies and otherantibody-like molecules that lack antigen specificity. Naturalantibodies and immunoglobulins are generally heterotetramers of about150,000 Daltons consisting of 2 identical light chains (L chains) and 2identical heavy chains (H chains). Each of the light chain is connectedto a heavy chain through a single covalent disulfide bond. However, thenumber of disulfide bonds between the heavy chains varies depending onthe isotype of the immunoglobulin. Both of the heavy and light chainsfurther have intramolecular disulfide bridges at constant distance. Eachof the heavy chain has a variable region (V_(H)) at one end and manyconstant regions connected thereto. Each of the light chain has avariable region (V_(L)) at one end and a constant region at the otherend. The constant region and the variable region of the light chain areplaced side-by-side to the first constant region and the variable regionof the heavy chain, respectively. Specific amino acid residues areconsidered to form the interface of the variable region of the light andheavy chains (Chothia C. et al. J. Mol. Biol. 186: 651-663, 1985;Novotny J., Haber E., Proc. Natl. Acad. Sci. USA 82: 4592-4596, 1985).

The light chains of antibodies (immunoglobulins) derived from vertebratespecies can be divided into two clearly distinct types called kappa (κ)and lambda (λ), based on the amino acid sequence of the constant region.In addition, an “immunoglobulin” can be classified into differentclasses based on the amino acid sequence of the constant region of theheavy chain. At least five major classes exist for immunoglobulins: IgA,IgD, IgE, IgG and IgM. Furthermore, some of them can be furtherclassified into subclasses (isotypes), for example, IgG-1, IgG-2, IgG-3and IgG-4, and IgA-1 and IgA-2. The heavy chain constant regions of thedifferent classes are called α, δ, ε, γ and μ, respectively. The subunitstructures and three-dimensional structures of immunoglobulins of eachclass are well known.

Herein, the phrase “monoclonal antibody” refers to an antibody obtainedfrom a group of substantially homogeneous antibodies, i.e., an antibodygroup wherein the antibodies constituting the group are homogeneousexcept for naturally occurring mutants that exist in a small amount. Amonoclonal antibody is highly specific and interacts with a singleantigenic site. Furthermore, each monoclonal antibody targets a singleantigenic determinant (epitope) on an antigen, as compared to common(polyclonal) antibody preparations that typically contain variousantibodies against diverse antigenic determinants. In addition to theirspecificity, monoclonal antibodies are advantageous in that they areproduced from hybridoma cultures not contaminated with otherimmunoglobulins.

The qualifier “monoclonal” indicates the characteristics of antibodiesobtained from a substantially homogeneous group of antibodies, and doesnot require that the antibodies be produced by a particular method. Forexample, the monoclonal antibody used in the present invention can beproduced by, for example, the hybridoma method (Kohler G. and MilsteinC., Nature 256: 495-497, 1975) or the recombination method (U.S. Pat.No. 4,816,567). The monoclonal antibodies used in the present inventioncan be also isolated from a phage antibody library (Clackson T. et al.,Nature 352: 624-628, 1991; Marks J. D. et al., J. Mol. Biol.222:581-597, 1991). The monoclonal antibodies in the presentspecification particularly include “chimeric” antibodies(immunoglobulins) wherein a part of the heavy chain and/or light chainis derived from a specific species, or a specific antibody class orsubclass and the remaining portion of the chain from another species, oranother antibody class or subclass. Furthermore, as long as they havethe desired biological activity, antibody fragments thereof are alsoincluded in the present invention (U.S. Pat. No. 4,816,567; Morrison S.L. et al., Proc. Natl. Acad. Sci. USA 81: 6851-6855, 1984).

The phrase “mutant antibody” refers to amino acid sequence variants ofantibodies wherein one or more amino acid residues are altered. The“mutant antibody” herein includes variously altered amino acid variantsas long as they have the same binding specificity as the originalantibody. Such mutants have less than 100% homology or similarity to theamino acid sequence that has at least 75%, more preferably at least 80%,even more preferably at least 85%, still more preferably at least 90%,and most preferably at least 95% amino acid sequence homology orsimilarity to the amino acid sequence of the variable region of theheavy chain or light chain of an antibody. The method of the presentinvention is equally applicable to both polypeptides, antibodies andantibody fragments; therefore, these terms are often usedinterchangeably.

The phrase “antibody fragment” refers to a part of a full-lengthantibody and generally indicates an antigen-binding region or a variableregion. For example, antibody fragments include Fab, Fab′, F(ab′)₂ andFv fragments. Papain digestion of an antibody produces two identicalantigen-binding fragments called Fab fragments each having anantigen-binding region, and a remaining fragment called “Fc” since itcrystallizes easily. On the other hand, by the digestion with pepsin, aF(ab′)₂ fragment (which has two antigen-binding sites and can cross bindantigens) and the remaining other fragment (called pFc′) are obtained.Other fragments include diabody (diabodies), linear antibodies,single-chain antibodies, and multispecific antibodies formed fromantibody fragments. In this specification, “functional fragment” of anantibody indicates Fv, F(ab) and F(ab′)₂ fragments.

Herein, an “Fv” fragment is the smallest antibody fragment and containsa complete antigen recognition site and a binding site. This region is adimmer (V_(H)-V_(L) dimmer) wherein the variable regions of each of theheavy chain and light chain are strongly connected by a noncovalentbond. The three CDRs of each of the variable regions interact with eachother to form an antigen-binding site on the surface of the V_(H)-V_(L)dimmer. Six CDRs confer the antigen-binding site of an antibody.However, a variable region (or a half of Fv which contains only threeCDRs specific to an antigen) alone has also the ability to recognize andbind an antigen although its affinity is lower than the affinity of theentire binding site.

Moreover, a Fab fragment (also referred to as F(ab)) further includesthe constant region of the light chain and a constant region (C_(H)1) ofthe heavy chain. An Fab′ fragment differs from the Fab fragment in thatit additionally has several residues derived from the carboxyl end ofthe heavy chain C_(H)1 region which contains one or more cysteines fromthe hinge domain of the antibody. Fab′-SH indicates an Fab′ wherein oneor more cysteine residues of the constant region has a free thiol-group.The F(ab′) fragment is produced by the cleavage of disulfide bondsbetween the cystines in the hinge region of the F(ab′)₂ pepsin digest.Other chemically bound antibody fragments are also known by thoseskilled in art.

The term “diabody (diabodies)” refers to a small antibody fragmenthaving two antigen-binding sites, and the fragment contains V_(H)-V_(L)wherein the heavy chain variable region (V_(H)) is connected to thelight chain variable region (V_(L)) in the same polypeptide chain. Whena short linker is used between the two regions so that the two regionscannot be connected together in the same chain, these two regions formpairs with the constant regions in another chain to create twoantigen-binding sites. The diabody is described in detail in, forexample, European patent No. 404,097, WO 93/11.161 and Holliger P. etal. (Proc. Natl. Acad. Sci. USA 90: 6444-6448, 1993).

A single-chain antibody (hereafter also referred to as single-chain Fvor sFv) or sFv antibody fragment contains the V_(H) and V_(L) regions ofan antibody, and these regions exist on a single polypeptide chain.Generally, an Fv polypeptide further contains a polypeptide linkerbetween the V_(H) and V_(L) regions, and therefore an sFv can form astructure necessary for antigen binding. See, Pluckthun “ThePharmacology of Monoclonal Antibodies” Vol. 113 (Rosenburg and Mooreeds. (Springer Verlag, New York) pp. 269-315, 1994) for the review ofsFv.

A multispecific antibody is an antibody that has specificity to at leasttwo different kinds of antigens. Although such a molecule usually bindsto two antigens (i.e., a bispecific antibody), the “multispecificantibody” herein encompasses antibodies that has specificity to morethan two antigens (for example, three antigens). The multispecificantibody can be a full-length antibody or fragments thereof (forexample, F(ab′)₂ bispecific antibody).

The phrase “humanized antibody” in the present invention is an antibodyproduced by genetic engineering. Specifically, it refers to an antibodycharacterized by a structure wherein a part of or the entire CDR of thehypervariable region is derived from that of a monoclonal antibody of anon-human mammal (mouse, rat, hamster, etc.), and the framework regionof the variable region and constant region are those derived from humanimmunoglobulin. Herein, the CDR of a hypervariable region refers to thethree regions (CDR1, CDR2 and CDR3) directly binding to an antigen in acomplementary manner and that exist in the hypervariable region of thevariable region of an antibody. Whereas, the framework region of avariable region refers to the relatively conserved four regions(framework regions; FR1, FR2, FR3 and FR4) which intervene between thethree above-mentioned CDR regions. Specifically, the “humanizedantibody” in the present invention refers to antibodies wherein allregions except a part or the entire CDR of the hypervariable region of amonoclonal antibody derived from a non-human mammal is replaced with acorresponding region of a human immunoglobulin.

Furthermore, a humanized antibody may contain residues that do not existin either the recipient antibody or the introduced CDR or the frameworksequence. Such alterations are performed to precisely optimize thecapability of the antibody. Generally, all humanized antibodiesessentially contain at least one, typically two variable regions. In theantibody, all or essentially all of the CDR regions correspond to theCDR of a non-human immunoglobulin, and all or essentially all of the FRsare derived from a human immunoglobulin variable region. Optimally, thehumanized antibody further may contain typically at least a part of theconstant region of a human immunoglobulin. More details can be found inJones P. T. et al. (Nature 321: 522-525, 1986), Riechmann L. et al.(Nature 332: 323-327, 1988) and Presta et al. (Curr. Op. Struct. Biol.2: 593-596, 1992).

The term “variable” in the antibody variable region indicates that acertain region in the variable region highly varies among antibodies,and that the region is responsible for the binding and specificity ofrespective antibodies to their specific antigens. The variable regionsare concentrated in three areas called CDR or hypervariable regionwithin the variable regions of light and heavy chains. There are atleast two methods to determine the CDR: (1) a technique based onsequence variation among species (i.e., Kabat et al., “Sequence ofProteins of Immunological Interest” (National Institute of Health,Bethesda) 1987); and (2) a technique based on crystallographic researchof antigen-antibody complex (Chothia C. et al., Nature 342: 877-883,1989). The area more highly conserved in the variable region is calledFR. The variable regions of natural heavy and light chains mainly haveβ-sheet structures and form three loop-like connections, and in somecases, contain four FRs connected by CDRs that form a β-sheet structure.The CDRs in each chain is maintained very closely to the CDRs on theother chain by FRs and plays a role in the formation of theantigen-binding site of an antibody (see, Kabat et al.). The constantregion does not directly participate in the binding of the antibody tothe antigen. However, it shows various effector functions, such asparticipation of the antibody in antibody dependent cytotoxicity.

The constant region of a human immunoglobulin has a unique amino acidsequences for each isotype, such as IgG (IgG1, IgG2, IgG3 and IgG4),IgM, IgA, IgD and IgE. In the present invention, the constant region ofthe above-mentioned humanized antibody may be of any isotype.Preferably, the constant region of human IgG is used. Moreover, there isno limitation on the FR of the variable region derived from a humanimmunoglobulin.

The term “antigen” in the present specification encompasses bothcomplete antigens having immunogenicity and incomplete antigens(including haptens) without immunogenicity. Antigens include substancessuch as proteins, polypeptides, polysaccharides, nucleic acids andlipids; however, they are not limited thereto. As immunogens forantibody production, soluble antigens or fragments thereof connected toother molecules may be used. In the interest of transmembrane molecules,such as receptors, fragments thereof (for example, extracellular regionsof receptors) may be used as immunogens. Alternatively, cells expressingtransmembrane molecules may be used as immunogens. Such cells may benatural cells (for example, tumor cell lines) or cells transfected byrecombinant techniques to express the transmembrane molecules. Any formof antigen known to those skilled in the art can be used to produceantibodies.

Herein, the phrase “antigen-binding protein” refers to proteins thathave the ability to bind to an antigen.

The phrase “immunoglobulin superfamily” in the present specificationrefers to proteins that have the structural characteristic wherein oneor multiple domains homologous to the constant or variable domain of animmunoglobulin are contained. The immunoglobulin superfamily includesthe immunoglobulin (H chain and L chain), T cell receptor (α chain, βchain, γ chain and δ chain), MHC class I molecule (α chain), β₂microglobulin, MHC class II molecule (α chain and β chain), CD3 (γchain, δ chain and ε chain), CD4, CD8 (α chain and β chain), CD2, CD28,LFA-3, ICAM-1, ICAM-2, VCAM-1, PECAM-1, F_(c) receptor II, poly Igreceptor, Thy-1, NCAM, myelin-associated glycoprotein (MAG), Po,carcinoembryonic antigen (CEA), PDGF receptor and so on.

The phrase “pharmaceutical agent” in the present specification refers tosubstances that are administered to animals for purposes such astreatment or prevention of diseases, injuries and such, or improvementof health conditions.

1. Amino Acid Alternation for Protein Stabilization

The present invention provides a method for stabilizing a proteinwherein an amino acid adjacent to an amino acid being deamidated in theprotein is substituted with another amino acid. The protein to bestabilized according to the present invention is not restricted in anyway. A suitable example of the protein includes antibodies. Humanizedantibodies or human antibodies are preferred as the antibody from theaspect of medical use.

In addition to asparagine, glutamine is also known as an amino acid thatis deamidated (Scotchler J. W. and Robinson A. B., Anal. Biochem. 59:319-322, 1974). When comparing peptides of 5 amino acids, the half-lifeof glutamine is 96 to 3409 days compared to the half-life of asparaginebeing 6 to 507 days. Namely, the reaction rate of deamidation ofglutamine is very slow compared with that of asparagine (Bischoff R. andKolbe H. V. J., J. Chromatogr. B. 662: 261-278, 1994). Deamidation ofglutamine has not been detected in antibody preparations (Harris R. J.,Kabakoff B., Macchi F. D., Shen F. J., Kwong M., Andya J. D. et al., J.Chromatogr. B. 752: 233-245, 2001). However, the deamidation reaction issupposed to be enhanced in vivo than in pharmaceutical preparations(Robinson N. E. and Robinson A. B., Proc. Natl. Acad. Sci. USA 98:12409-12413, 2001). Therefore, to develop an antibody preparation with along in vivo half-life, suppression of deamidation of glutamine, inaddition to asparagine is considered to be necessary. The amino acid tobe deamidated preferably is asparagine.

Amino acids other than glycine can be also considered as the amino acidadjacent to an deamidated amino acid and that can be substituted in aprotein (Robinson N. E. and Robinson A. B., Proc. Natl. Acad. Sci. USA98: 4367-4372, 2001). However, glycine is particularly known to causedeamidation of asparagine. Thus, the amino acid that is located adjacentto an amino acid that is deamidated preferably is glycine.

Generally, an antibody is inactivated by amino acid substitution in theCDR. However, the present inventors revealed that the activity of anantibody is retained even after the substitution of an amino acidadjacent to asparagine in the CDR, and hence the stability of theantibody can be improved. Therefore, according to the present invention,an amino acid adjacent to an asparagine in the CDR is effectivelysubstituted with another amino acid. Glycine is a suitable target as theamino acid adjacent to the asparagine. Particularly, glycine containedin the “Asn-Gly” sequence that is particularly easily deamidated is themost suitable target.

According to the present invention, in addition to the amino acidadjacent to the above-mentioned deamidated amino acid, one or more ofother amino acids can also be altered unless the stability andbiological activity of the protein is reduced. When the protein is anantibody, biological activity indicates its activity to specificallybind to antigen. A preferred amino acid alternation is a conservativesubstitution from the viewpoint to maintain the property of the protein.

The alteration of an amino acid of a protein can be performed by methodsto recombine the gene sequence encoding the protein. Techniquesgenerally known in the art can be used for gene recombination.

When the protein is an antibody, the alteration of amino acids can beperformed as follows. For example, variant antibodies or mutants whereinone or more amino acid residues are altered in one or more of thehypervariable regions of the antibody can be prepared. In addition, oneor more mutations (for example, substitution) can be introduced into theframework residues of the mammalian antibody to improve the bindingaffinity of the mutant antibody to its antigen. Exemplary frameworkresidues that can be altered include portions that directly bind toantigens by noncovalent bonds (Amit A. G. et al., Science 233: 747-753,1986), portions that affect and/or influence the structure of the CDR(Chothia C. and Lesk A. M., J. Mol. Biol. 196: 901-917, 1987) and/orportions that are involved in the VL-VH interaction (European patent No.239,400, B1). According to an embodiment, the binding affinity of anantibody to an antigen is enhanced by altering one or more of suchframework residues.

One useful method for producing mutant antibodiesis “Alanine-ScanningMutagenesis” (Cunningham B. C. and Wells J. A., Science 244: 1081-1085,1989; Cunningham B. C. and Wells J. A., Proc. Natl. Acad. Sci. USA 84:6434-6437, 1991). According to this method, one or more residues of thehypervariable region are substituted with alanine or polyalanineresidues to change the interaction between the antigen and thecorresponding amino acids. The residues of the hypervariable region thatshowed functional sensitivity to the substitution are furtherdistinguished in more detail by introducing further or other mutation tothe substitution site. Therefore, although the site to introduce anamino acid sequence mutation is determined beforehand, the type ofmutation does not have to be determined beforehand.

The ala mutant produced by this method is screened for its biologicalactivity. Depending on the desired characteristics obtained by thescanned residues, a similar substitution of other amino acids may alsobe performed. Alternatively, there is also a method wherein the alteredamino acid residue is more systematically identified. According to thismethod, the hypervariable region residues within a species-specificantibody involved in the binding of a first mammalian species antigenand the hypervariable region residues involved in the binding of ahomologous antigen of a second mammalian species can be identified. Inorder to achieve this, Alanine-scanning is performed for thehypervariable region residues of the species-specific antibody. In thescanning, the binding of each ala mutant to the first and secondmammalian species antigen is tested in order to identify (1) thehypervariable region residues involved in the binding of the firstmammalian species (for example, human) antigen and (2) the site involvedin the binding of the second mammalian species (for example, non-human)antigen homolog. Preferably, residues that are apparently involved inthe binding of the second mammalian species (for example, non-humanmammalian) derived-antigen but not in the binding of the first mammalianspecies (for example, human) derived-antigen are candidates foralteration. In another embodiment, residues that are clearly involved inthe binding of the first and second mammalian species derived-antigensare selected for alteration. The alteration includes deletion of theresidues and insertion wherein one or more residues are linked to thetarget residues; however, generally, alteration refers to substitutionof the residues with other amino acids.

A nucleic acid molecule encoding an amino acid sequence mutant may beprepared by various methods known in the art. Such methods include, butare not limited to, oligo nucleotide mediated mutation (or site-specificmutation), PCR mutation or cassette mutation of a previously producedmutated or a non-mutated version of a species-specific antibody.Suitable methods for producing mutants include site-specific mutation(see Kunkel T. A., Proc. Natl. Acad. Sci. USA 82: 488-492, 1985) andsuch. Generally, mutant antibodies having improved biologicalcharacteristics have at least 75%, preferably at least 80%, morepreferably at least 85%, further more preferably at least 90% and mostpreferably at least 95% amino acid sequence homology or similarity withthe amino acid sequence of the variable region of the heavy or lightchain of the original antibody. Sequence homology or similarity in thepresent specification is defined as the rate of the amino acid residueswhich are homologous (i.e., the same residues) or similar (i.e., theamino acid residues of the same group based on the above-mentionedgeneral side chain characteristic) to the residues in the speciesspecific antibody of the candidate sequence after alignment of thesequence and introducing a gap as needed in order to obtain the maximumsequence homology.

Alternatively, a mutant antibody can be constructed by systematicmutations of the CDR in the heavy and light chains of an antibody.Preferable methods for constructing such a mutant antibody includemethods utilizing affinity maturation using phage display (Hawkins R. E.et al, J. Mol. Biol. 226: 889-896, 1992; Lowman H. B. et al,Biochemistry 30: 10832-10838, 1991). Bacteriophage coat protein fusion(Smith G. P., Science 228:1315-1317, 1985; Scott J. K. and Smith G. P.,Science 249: 386-390, 1990; Cwirla S. E. et al., Proc. Natl. Acad. Sci.USA 87: 6378-6382, 1990; Devlin J. J. et al., Science 249: 404-406,1990; review by Wells and Lowman, Curr. Opin. Struct. Biol. 2:597, 1992;U.S. Pat. No. 5,223,409) is known as a useful method to relate adisplayed phenotype protein or peptide with the genotype of thebacteriophage particle encoding it. Moreover, a method to display theF(ab) region of an antibody on the surface of a phage is also known inthe art (McCafferty et al., Nature 348:552, 1990; Barbas et al., Proc.Natl. Acad. Sci. USA 88:7978, 1991; Garrard et al., Biotechnology9:1373, 1991). Monovalent phage display comprises the step of displayinga group of protein variants as fusions with a coat protein of thebacteriophage yet that only one copy of the variant is displayed on afew phage particles (Bass et al, Proteins 8:309, 1990).

Affinity maturation or improvement of equilibrium of the bindingaffinity of various proteins has been performed hitherto by mutagenesis,monovalent phage display, functional analysis and addition of desirablemutations of, for example, human growth hormone (Lowman and Wells, J.Mol. Biol. 234: 564-578, 1993; U.S. Pat. No. 5,534,617) and antibodyF(ab) region (Barbas et al., Proc. Natl. Acad. Sci. USA 91:3809, 1994;Yang et al., J. Mol. Biol. 254:392, 1995). A library of many proteinvariants (10⁶ molecules) different at specific sequence sites can beprepared on the surface of bacteriophage particles that contain DNAsencoding specific protein variants. The displayed amino acid sequencecan be predicted from DNA by several cycles of affinity purificationusing immobilized antigen followed by isolation of respectivebacteriophage clones.

2. Production of Polyclonal Antibodies

Polyclonal antibodies are preferably produced in non-human mammals bymultiple subcutaneous (sc) or intraperitoneal (ip) injections of relatedantigen and adjuvant. The related antigen may be bound to a protein thatis immunogenic to the immunized species, for example, keyhole limpethemocyanin, serum albumin, bovine thyroglobulin or soybean trypsininhibitor, using bifunctional agents or inducers, for example,maleimidebenzoylsulfosuccinimide ester (binding via a cysteine residue),N-hydroxysuccinimide (via a lysine residue), glutaraldehyde, succinicanhydride, thionylchloride or R¹N═C═CR (wherein, R and R¹ are differentalkyl groups).

For example, an animal is immunized against an antigen, an immunogenicconjugate or a derivative through multiple endermic injections ofsolution containing 100 μg or 5 μg of protein or conjugate (amount for arabbit or a mouse, respectively) with 3 volumes of Freund's completeadjuvant. One month later, a booster is applied to the animal throughsubcutaneous injections of ⅕ to 1/10 volume of the original peptide orconjugate in Freund's complete adjuvant at several sites. Blood iscollected from the animal after 7 to 14 days and serum is analyzed forantibody titer. Preferably, a conjugate of the same antigen but that isbound to a different protein and/or bound via a different cross-linkingreagent is used as the booster. A conjugate can be also produced byprotein fusion through recombinant cell culture. Moreover, in order toenhance immune response, agglutinins, such as alum, are preferably used.The selected mammalian antibody usually has a binding affinity strongenough to the antigen. The affinity of an antibody can be determined bysaturation bonding, enzyme-linked immunosorbent assay (ELISA) andcompetitive analysis (for example, radioimmunoassay).

As a method of screening for desirable polyclonal antibodies,conventional cross-linking analysis described in “Antibodies, ALaboratory Manual” (Harlow and David Lane eds., Cold Spring HarborLaboratory, 1988) can be performed. Alternatively, for example, epitopemapping (Champe et al., J. Biol. Chem. 270: 1388-1394, 1995) may beperformed. Preferred methods for measuring the efficacy of a polypeptideor antibody include a method using the quantitation of the antibodybinding affinity. Other embodiments include methods wherein one or moreof the biological properties of an antibody are evaluated instead of theantibody binding affinity. These analytical methods are particularlyuseful in that they indicate the therapeutic efficacy of an antibody.Antibodies that show improved properties through such analysis have alsogenerally, but not always, enhanced binding affinity.

3. Production of Monoclonal Antibodies

A monoclonal antibody is an antibody that recognizes a single antigensite. Due to its uniform specificity, a monoclonal antibody is generallyuseful than a polyclonal antibody which contains antibodies recognizingmany different antigen sites. A monoclonal antibody can be produced bythe hybridoma method (Kohler et al., Nature 256:495, 1975), therecombinant DNA method (U.S. Pat. No. 4,816,567), and so on.

According to the hybridoma method, a suitable host animal, such asmouse, hamster or rhesus monkey, is immunized similar as described aboveto produce antibodies that specifically bind to a protein used forimmunization or to induce lymphocytes producing the antibodies.Alternatively, a lymphocyte may be immunized in vitro. Then, thelymphocyte is fused with a myeloma cell using suitable fusion agents,such as polyethylene glycol, to generate a hybridoma cell (Goding,“Monoclonal Antibodies: Principals and Practice”, Academic Press, pp.59-103, 1986). Preferably, the produced hybridoma cell is seeded andcultured on a proper culture media containing one or more substancesthat inhibit proliferation or growth of unfused parental myeloma cells.For example, when the parental myeloma cell lacks the hypoxantin guaninephosphoribosyl transferase enzyme (HGPRT or HPRT), the culture media forthe hybridoma typically contains substances that inhibit the growth ofHGRPT deficient cells, i.e., hypoxantin, aminopterin and thymidine (HATculture media).

Preferred myeloma cells include those that can efficiently fuse, produceantibodies at a stable high level in selected antibody producing cells,and are sensitive to media such as HAT media. Among the myeloma celllines, preferred myeloma cell lines include mouse myeloma cell lines,such as mouse tumor derived cells MOPC-21 and MPC-11 (obtained from SalkInstitute Cell Distribution Center, San Diego, USA), and SP-2 andX63-Ag8-653 cells (obtained from the American Type Culture Collection,Rockville, USA). Human myeloma and mouse-human heteromycloma cell lineshave also been used for the production of human monoclonal antibodies(Kozbar, J. Immunol. 133:3001, 1984; Brodeur et al., “MonoclonalAntibody Production Techniques and Application”, Marcel Dekker Inc, NewYork, pp. 51-63, 1987).

Next, the production of monoclonal antibodies against an antigen in theculture media wherein the hybridoma cells had been cultured is analyzed.Preferably, the binding specificity of the monoclonal antibody producedfrom the hybridoma cells is measured by in vitro binding assay, such asimmunoprecipitation, radioimmunoassay (RIA) or enzyme-linkedimmunosorbent assay (ELISA). After identifying the hybridoma cells thatproduce antibodies having the desired specificity, affinity and/oractivity, clones are subcloned by limiting dilution method and culturedby standard protocols (Goding, “Monoclonal Antibodies: Principals anPractice”, Academic Press, pp. 59-103, 1986). Culture media suitable forthis purpose include, for example, DMEM and RPMI-1640. Furthermore, ahybridoma cell can also be grown as ascites tumor in an animal in vivo.Monoclonal antibodies secreted from a subclone are preferably purifiedfrom culture media, ascites or serum via conventional immunoglobulinpurification methods, such as protein A-Sepharose, hydroxyapatitechromatography, gel electrophoresis, dialysis or affinitychromatography.

DNA encoding a monoclonal antibody can be easily isolated and sequencedby conventional methods, for example, using an oligo nucleotide probespecifically binding to genes encoding the heavy and light chains of themonoclonal antibody. Hybridoma cells are preferred starting materialsfor obtaining such DNAs. Once the DNA is isolated, it is inserted intoan expression vector and transformed into a host cell, such as E. colicell, simian COS cell, Chinese hamster ovary (CHO) cell or myeloma cell,that produce no immunoglobulin protein unless being transformed, andmonoclonal antibody is produced from the recombinant host cell. Inanother embodiment, an antibody or an antibody fragment can be isolatedfrom an antibody phage library prepared by the method described byMcCafferty et al. (Nature 348: 552-554, 1990). Clackson et al. (Nature352: 624-628, 1991) and Marks et al. (J. Mol. Biol. 222: 581-597, 1991)describe the isolation of mouse and human antibodies using phagelibraries, respectively. The following references describe theproduction of high affinity (nM range) human antibody by chain shuffling(Marks et al, Bio/Technology 10: 779-783, 1992), and combinatorialinfection and in vivo recombination for producing large phage libraries(Waterhouse et al, Nucl. Acids Res. 21: 2265-2266, 1993). Thesetechniques can also be used to isolate monoclonal antibodies in place ofconventional monoclonal antibody hybridoma techniques.

DNA can be also altered by, for example, substitution of correspondingmouse sequences with the coding sequences of the constant regions ofhuman heavy and light chains (U.S. Pat. No. 4,816,567; Morrison et al,Proc. Natl. Acad. Sci. USA 81:6851, 1984), or by binding immunoglobulinpolypeptides through covalent bonds. Typically, these non-immunoglobulinpolypeptides are substituted with the constant region of an antibody orthe variable region of the antibody antigen-binding site is substitutedto construct a chimeric bispecific antibody that has an antigen-bindingsite specific for an antigen and another antigen-binding site specificfor another antigen.

4. Production of Antibody Fragments

Hitherto, antibody fragments have been produced by digesting naturalantibody with proteases (Morimoto et al., J. Biochem. Biophys. Methods24: 107-117, 1992; Brennan et al., Science 229:81, 1985). However,today, they can also be produced by recombinant techniques. For example,antibody fragments can also be isolated from the above-mentionedantibody phage library. Furthermore, F(ab′)₂-SH fragments can bedirectly collected from a host cell such as E. coli, and chemicallybound in the form of F(ab′)₂ fragment (Carter, et al., Bio/Technology10: 163-167, 1992). Moreover, in another method, F(ab′)₂ fragment canalso be directly isolated from recombinant host cell culture. The methodfor constructing single chain antibodies, fragments of single chainantibodies and such are well known in the art (for example, see, U.S.Pat. No. 4,946,778; U.S. Pat. No. 5,260,203; U.S. Pat. No. 5,091,513;U.S. Pat. No. 5,455,030; etc.).

5. Production of Multispecific Antibodies

Methods for producing multispecific antibodies are known in the art. Theproduction of a full-length bispecific antibody includes the step ofco-expression of two immunoglobulin heavy-light chains having differentspecificity (Millstein et al., Nature 305: 537-539, 1983). The heavy andlight chains of immunoglobulins are randomly combined, and therefore,the obtained multiple co-expressing hybridomas (quadroma) are a mixtureof hybridomas each expressing a different antibody molecule. Thus, thehybridoma producing the correct bispecificity antibody has to beselected among them. The selection can be performed by methods such asaffinity chromatography. Furthermore, according to another method, thevariable region of an antibody having the desired binding specificity isfused to the constant region sequence of an immunoglobulin. Theabove-mentioned constant region sequence preferably contains at least apart of the hinge, CH2 and the CH3 regions of the heavy chain constantregion of the immunoglobulin. Preferably, the CH1 region of the heavychain required for the binding with the light chain is further included.DNA encoding the immunoglobulin heavy chain fusion is inserted into anexpression vector to transform a proper host organism. If needed, DNAencoding the immunoglobulin light chain is also inserted into anexpression vector different to that of the immunoglobulin heavy chainfusion to transform the host organism. There are cases where theantibody yield increases when the ratio of the chains is not identical.In such cases, it is more convenient to insert each of the genes intoseparate vectors since the expression ratio of each of the chains can becontrolled. However, genes encoding plural chains can also be insertedinto a vector.

According to a preferred embodiment, a bispecific antibody is desiredwherein a heavy chain having a first binding specificity exists as anarm of the hybrid immunoglobulin and a heavy chain-light chain complexhaving another binding specificity exists as the other arm. Due to theexistence of the light chain only on one of the arms, the bispecificantibody can be readily isolated from other immunoglobulins. Such aseparation method is referred to in WO 94/04690. See, Suresh et al.(Methods in Enzymology 121:210, 1986) for further reference of methodsfor producing bispecific antibodies. A method wherein a pocketcorresponding to a bulky side chain of a first antibody molecule iscreated in a multispecific antibody that comprises the antibody constantregion CH3 (WO 96/27011) is also known as a method for decreasinghomodimers to increase the ratio of heterodimers in the final productobtained from recombinant cell culture. According to the method, one ofthe antibody molecules is altered at one or more amino acids on thesurface that binds to the other molecule to amino acids having a bulkyside chain (e.g., tyrosine or tryptophan). Furthermore, amino acids witha bulky side chain in the corresponding portion of the other antibodymolecule is altered to amino acids with a small side chain (e.g.,alanine or threonine).

Bispecific antibodies include, for example, heteroconjugated antibodieswherein one antibody is bound to avidin and the other to biotin and such(U.S. Pat. No. 4,676,980, WO 91/00360, WO 92/00373, European patent No.03089). Cross-linkers used for the production of such heteroconjugatedantibodies are well known, and are mentioned, for example in U.S. Pat.No. 4,676,980.

Additionally, methods for producing bispecific antibodies from antibodyfragments have been also reported. For example, bispecific antibodiescan be produced utilizing chemical bonds. For example, first, F(ab′)₂fragments are produced and the fragments are reduced in the presence ofdithiol complexing agent, sodium arsanilate, to prevent intramoleculardisulfide formation. Next, the F(ab′)₂ fragments are converted tothionitrobenzoate (TNB) derivatives. After re-reducing one of theF(ab′)₂-TNB derivatives to a Fab′-thiol using mercaptoethanolamine,equivalent amounts of the F(ab′)₂-TNB derivative and Fab′-thiol aremixed to produce a bispecific antibody.

Various methods have been reported to directly produce and isolatebispecific antibodies from recombinant cell culture. For example, aproduction method for bispecific antibodies using a leucine zipper hasbeen reported (Kostelny et al., J. Immunol. 148:1547-1553, 1992). First,leucine zipper peptides of Fos and Jun proteins are connected to theFab′ sites of different antibodies by gene fusion, the homodimerantibodies are reduced at the hinge region to form monomers, followed byreoxidation to form a heterodimer antibody. Alternatively, a method toform two antigen-binding sites wherein pairs are formed betweendifferent complementary light chain variable regions (VL) and heavychain variable regions (VH) by linking the VL and VH through a linkerthat is short enough to prevent binding between these two regions(Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448, 1993).Furthermore, dimers utilizing single chain Fv (sFV) have also beenreported (Gruger et al., J. Immunol. 152:5368, 1994). Moreover,trispecific (rather than bispecific) antibodies have also been reported(Tutt et al., J. Immunol. 147:60, 1991).

6. Production of Humanized Antibodies

Humanized antibodies can be obtained via established general antibodyproduction methods by immunizing a human antibody producing transgenicnon-human mammal with an immunogen (antigen). Methods for producinghuman antibody producing non-human mammals, particularly human antibodyproducing transgenic mice, are known in the art (Nature Genetics 7:13-21, 1994; Nature Genetics 15: 146-156, 1997; Published JapaneseTranslation of International Publication No. Hei 4-504365; PublishedJapanese Translation of International Publication No. Hei 7-509137;Nikkei Science 6: 40-50, 1995; WO 94/25585; Nature 368: 856-859, 1994;Published Japanese Translation of International Publication No. Hei6-500233; etc.). Specifically, the human antibody producing transgenicnon-human mammal can be produced by the following steps:

-   (1) producing a knockout non-human mammal wherein the endogenous    immunoglobulin heavy chain gene of the animal is functionally    inactivated via the substitution of at least a part of the    endogenous immunoglobulin heavy chain locus of the non-human mammal    with a drug resistance marker gene (for example, neomycin resistance    gene) by homologous recombination;-   (2) producing a knockout non-human mammal wherein the endogenous    immunoglobulin light chain gene (particularly, the κ chain gene) of    the animal is functionally inactivated via the substitution of at    least a part of the endogenous immunoglobulin light chain locus of    the non-human mammal with a drug resistance marker gene (for    example, neomycin resistance gene) by homologous recombination;-   (3) producing a transgenic non-human mammal wherein a desired region    of the human immunoglobulin heavy chain locus has been integrated    into the mouse chromosome using a vector represented by yeast    artificial chromosome (YAC) vector and such that can transfer large    genes;-   (4) producing a transgenic non-human mammal wherein a desired region    of the human immunoglobulin light chain (particularly, the κ chain)    locus has been integrated into the mouse chromosome using a vector    represented by YAC vector and such that can transfer large genes;    and-   (5) producing a transgenic non-human mammal wherein both the    endogenous immunoglobulin heavy chain and light chain loci of the    non-human mammal are functionally inactivated, yet desired regions    of both the human immunoglobulin heavy chain and light chain are    integrated into the non-mammalian chromosome by crossing the    knockout non-human mammals and transgenic non-human mammals of    above-mentioned (1) to (4) in an arbitrary order.

As mentioned above, an endogenous immunoglobulin locus of non-humanmammals can be inactivated so that it inhibits reconstitution of thelocus via the substitution of a proper region of the locus with anexogenous marker gene (for example, neomycin resistance gene, etc.) viahomologous recombination. For inactivation using the homologousrecombination, for example, a method called positive negative selection(PNS) can be used (Nikkei Science 5: 52-62, 1994). The functionalinactivation of an immunoglobulin heavy chain locus can be attained by,for example, introducing a deficit into a part of the J or C region (forexample, the Cμ region). On the other hand, the functional inactivationof an immunoglobulin light chain (for example, the κ chain) can beattained by, for example, introducing a deficit into a part of the J orC region, or a region comprising the area that spans over both the J andC regions.

A transgenic animal can be produced by standard methods (for example,“Saishin-Dobutsusaibou-Jikken manual (The latest animal cell experimentmanual)”, Chapter 7, LIC, pp. 361-408, 1990). Specifically, ahypoxantin-guanine phosphoribosyltransferase (HRPT) negative embryonicstem (ES) cell derived from normal non-human animal blastocyst is fusedby the spheroplast fusion method with yeast that comprises a YAC vectorinserted with a gene or a part thereof encoding the human immunoglobulinheavy chain or light chain locus and the HRPT gene. The ES cell whereinthe exogenous gene has been integrated into the mouse endogenous gene isselected by HAT selection. Subsequently, the selected ES cell ismicroinjected into a fertilized egg (blastocyst) obtained from anothernormal non-human mammal (Proc. Natl. Acad. Sci. USA 77: 7380-7384, 1980;U.S. Pat. No. 4,873,191). A chimeric transgenic non-human mammal is bornby transplanting the blastocyst into the uterus of another non-humanmammal that acts as the surrogate mother. Heterotransgenic non-humanmammals are obtained by crossing the chimeric animal with a normalnon-human mammal. By crossing the heteroanimals among themselves,homotransgenic non-human mammals can be obtained according to Mendel'slaw.

A humanized antibody can also be obtained from the culture supernatantby culturing a recombinant human monoclonal antibody producing host thatcan be obtained via the transformation of the host with cDNAs encodingeach of the heavy and light chains of such humanized antibody orpreferably a vector containing the cDNAs by recombinant technique.Herein, such a host is a eukaryotic cell other than fertilized egg,preferably a mammalian cell, such as CHO cell, lymphocyte and myelomacell.

The antigen-binding activity of an antibody stabilized by the method ofthe present invention is not particularly limited; however, it ispreferred to have 70% or more, more preferably 80% or more and furtherpreferably 90% or more of the activity possessed by the antibody beforethe amino acid substitution.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the amino acid sequences of humanized heavy chain versioni and humanized light chain version b2 contained in the anti-human TFantibody described in WO 99/51743. A few asparagine residues (Asn28,Asn51 and Asn55) that may be deamidated are boxed.

FIG. 2 depicts the cloning vector pCVIDEC-AHi integrated with the heavychain variable region (AHi) of the anti-human TF antibody. A: the entirepCVIDEC-AHi vector; and B: the NheI-SalI fragment of the heavy chainvariable region.

FIG. 3 depicts the anion exchange chromatogram of each of the anti-humanTF mutant antibodies and the original anti-human TF antibody. A: 99D01;and B: original (native).

FIG. 4 depicts the anion exchange chromatogram of each of the anti-humanTF mutant antibodies and the original anti-human TF antibody. C: N28D;and D: N51D.

FIG. 5 depicts the anion exchange chromatogram of each of the anti-humanTF mutant antibodies and the original anti-human TF antibody. E: N54D;and F: N51D/N54D.

FIG. 6 depicts the superposed anion exchange chromatograms of each ofthe anti-human TF mutant antibodies and the original anti-human TFantibody.

FIG. 7 depicts the binding activity of each of the anti-human TF mutantantibodies and the original anti-human TF antibody.

FIG. 8 depicts the neutralizing activity of each of the anti-human TFmutant antibodies and the original anti-human TF antibody.

FIG. 9 depicts the cloning vector pCVIDEC-AHi integrated with the heavychain variable region (AHi) of the anti-human TF antibody. Thenucleotide sequences described in FIG. 9 are shown in SEQ ID NOs: 27 and28. A: the entire pCVIDEC-AHi vector; B: the XbaI-BalI fragment of theheavy chain variable region; and C: the XbaI-ApoI fragment of the heavychain variable region.

FIG. 10 depicts a graph comparing the binding activity of each of theanti-human TF mutant antibodies. The anti-human TF antibodyconcentration conversion values calculated based on the calibrationcurve plotted using the bulk anti-human TF antibody (Lot No. 00C01) areshown. Blank: 10% FCS-α-MEM culture medium; control: CHO cell culturesupernatant; G to P: each of the anti-human TF antibody heavy chainGly55 mutants.

FIG. 11 depicts the binding activity of each of the anti-human TF mutantantibodies mutants.

FIG. 12 depicts the elution profiles of each of the anti-human TF mutantantibodies and the bulk anti-human TF antibody (99D01).

FIG. 13 depicts graphs showing the binding activity before and after theaccelerated testing on each of the anti-human TF mutant antibodies andthe bulk anti-human TF antibody (99D01), and the ratio compared with theinitial value.

FIG. 14 depicts the anion chromatograph and neutralizing activity beforeand after the accelerated testing on each of the anti-human TF mutantantibodies and the bulk anti-human TF antibody (99D01). A: 99D01 (G55G);B: G55L; C: G55I; D: G55F; E: G55E; and F: G55K.

FIG. 15 depicts the anion chromatograph and neutralizing activity beforeand after the accelerated testing on each of the anti-human TF mutantantibodies and the bulk anti-human TF antibody (99D01). A: 99D01 (G55G);B: G55L; C: G55I; D: G55F; E: G55E; and F: G55K.

FIG. 16 depicts the neutralizing activity before and after theaccelerated testing on each of the anti-human TF mutant antibodies andthe bulk anti-human TF antibody (99D01).

FIG. 17 depicts the neutralizing activity before and after theaccelerated testing on each of the anti-human TF mutant antibodies andthe bulk anti-human TF antibody (99D01).

BEST MODE FOR CARRYING OUT THE INVENTION

Herein below, the present invention will be specifically described usingExamples; however, it is not to be construed as being limited thereto:

EXAMPLE 1 Measurement of Binding and Neutralizing Activities ofAnti-Human TF Antibody Asn54 Substitution Mutant with TF

Humanized antibody against human tissue factor (TF) described in WO99/51743 is expected to suppress thrombus formation without suppressingthe extrinsic blood coagulation reaction through the inhibition of theTF mediated Factor X activation in the intrinsic blood coagulationreaction. This anti-human TF antibody contains humanized heavy chainversion i (SEQ ID NO: 25, FIG. 1) and humanized light chain version b2(SEQ ID NO: 26, FIG. 1). The antibody comprises a few asparagineresidues that may be deamidated: such as Asn51 and Asn54 in CDR2 of theheavy chain variable region, and Asn28 in FR1 of the heavy chainvariable region. Particularly, Asn54 is contained in an Asn-Glysequence, and thus is considered to be easily deamidated.

Pharmaceutical formulation of the antibody has not been established.Under destabilizing conditions of the antibody, the antibody bindingactivity to TF decreases in a solution pH-dependent manner and theincrease of low pI molecular species has been observed. Due to theincrease of degeneration upon stronger basification, the decrease in thebinding activity and the increase of low pI molecular species aresupposed to result from the deamidation of amino acids constituting theanti-human TF antibody. Furthermore, the deamidation is suggested tooccur in the CDR region due to the co-observed decrease in the antigenbinding activity.

Based on these findings, mutants (4 mutants including N51D mutant, N54Dmutant, N51D/N54D double mutant and N28D mutant) wherein Asn51 and Asn54in the CDR2 and Asn28 in the FR1 of the heavy chain variable region ofanti-human TF antibody described in WO 99/51743 have been substitutedwith aspartic acid were prepared, and their binding activity andneutralizing activity to TF were measured.

The amino acid sequence of the antibody follows the sequence describedby Kabat et al. (Kabat E. A., Wu T. T., Perry H. M., Gottesman K. S. andFoeller C., “Sequences of proteins of immunological interest. 5th ed.”,US Dept. Health and Human Services, Bethesda, Md., 1991).

1. Construction of Anti-Human TF Mutant Antibody Expression Vector

Cloning vector pCVIDEC-AHi (FIG. 2A) and anti-human TF antibodyexpression vector pN5KG4P-AHi-ALb2 both integrated with the heavy chainvariable region (AHi) of the anti-human TF antibody were purified fromdam⁻/dcm⁻ E. coli SCS110.

Substitution of the codon encoding Asn with that of Asp was performed inpCVIDEC-AHi. Specifically, a fragment of about 30 bp containing theregion that encodes each Asn was digested with restriction enzymes andreplaced with a fragment prepared from a synthetic oligo DNA having basesubstitution (FIG. 2B). To alter Asn51 and Asn54, pCVIDE-AHi wasdigested with XbaI and BalI, and a fragment designed for one base pairsubstitution of the codon was integrated to alter either or both ofAsn51 and Asn54 in the heavy chain variable region CDR2 of theanti-human TF antibody to Asp. To alter Asn28, pCVIDEC-AHi was digestedwith MroI and EcoT22I, and a fragment designed for one base pairsubstitution of the codon was integrated to substitute Asn28 to Asp inthe heavy chain variable region FR1 of the anti-human TF antibody.

The sequence was confirmed at every step while constructing theexpression vector. The target sequence was confirmed on the cloningvector, and the sequence was reconfirmed after replacing the fragmentobtained by digestion with NheI and SalI with the heavy chain variableregion of the anti-human TF antibody expression vector digested withNheI and SalI. E. Coli DH5α was transformed after confirming that thetarget sequence was obtained. Then, the four anti-human TF mutantantibody expression vectors, i.e., N51D mutant expression vector, N54Dmutant expression vector, N51D/N54D double mutant expression vector andN28D mutant expression vector, were purified using the QIAGEN Maxicolumn.

2. Transient Expression of Anti-Human TF Mutant Antibody in COS-7 Cell

Five vectors in total, i.e., the constructed expression vectors for eachof the mutants and the original anti-human TF antibody, were transfectedinto COS-7 cells by the electroporation method and were transientlyexpressed. COS-7 cells were washed with D-PBS (−) and then resuspendedin PBS to be about 0.3 to 1.0×10⁷ cells/ml. The cell suspension wastransferred into a 0.4 cm cuvette together with 10 μg of anti-human TFmutant antibody expression vector, and electroporation was conductedwith the conditions of 1.5 kV and 25 μF. After leaving standing for 10min, the cells were suspended in 30 ml of 10% FCS-DMEM. On the next day,dead cells were discarded together with the media and 50 ml of fresh 10%FCS-DMEM was added. The cells were cultured for 3 days and then theculture supernatant was collected.

3. Measurement of Expression Level of Anti-Human TF Mutant Antibody

3-1 Measurement of Expression Level by Direct ELISA

100 μl each of the culture supernatant of the transfected COS-7 cellswere seeded on a 96-well ELISA plate and immobilized over night.Similarly, 100μ each of anti-human TF antibody (Lot No. 00C01) seriallydiluted (1 to 1000 ng/ml) with DMEM was seeded and immobilized on a96-well ELISA plate for plotting a calibration curve. After blockingwith ELISA dilution buffer, HRP-labeled anti-IgG antibody was reactedand color was developed by TMB. The reaction was quenched with 2 Msulfuric acid and the absorbance at 450 nm was measured with ARVO-SX5.The amount of anti-human TF antibody in the culture supernatant wascalculated from the value of the anti-human TF antibody (Lot No. 00C01)seeded for the calibration curve.

As shown in Table 1, direct ELISA confirmed concentration and totalexpression level of anti-TF antibody of about 65 to about 100 ng/ml andabout 3 to about 5 μg, respectively. TABLE 1 Total expressionConcentration Dosage level (ng/ml) (ml) (μg) Original 98.710 50 4.9 N28D84.535 50 4.2 N51D 75.634 50 3.8 N54D 77.956 50 3.9 N51D/N54D 68.387 503.44. Purification of Each Anti-Human TF Mutant Antibody

Each mutant was purified from 50 ml of the recovered culture supernatantusing affinity chromatography (Protein A) and anion exchangechromatography (Mono Q).

4-1 Affinity Chromatography

Affinity chromatography was performed under the following conditions:

-   -   System: SMART System (Amersham Pharmacia Biotech)    -   Column: HiTrap Protein A HP (0.7 cmφ×2.5 cm, 1 ml, Amersham        Pharmacia Biotech)    -   Equilibrating Buffer: D-PBS (−)    -   Washing Buffer: 10 mM Sodium phosphate buffer (pH 7.4)    -   Elution Buffer: 50 mM Acetic acid (pH 2 to 3)

After adjusting the pH to 7.4 with 0.5 M sodium monophosphate solution,a sample was concentrated 5-fold with Centriprep-50 and loaded at a flowrate of 1 ml/min onto the column equilibrated with 10 ml (10 C.V.)equilibrating buffer. The column was washed with 5 ml (5 C.V.) washingbuffer at a flow rate of 0.5 ml/min, eluted with 5 ml (5 C.V.) elutionbuffer, and then collected as ten fractions, each containing 0.5 mlsolution. Four fractions containing the antibody were combined andneutralized to pH 6 to 7 with 0.1 ml of 1 M Tris base.

4-2 Anion Exchange Chromatography

Next, anion exchange chromatography was performed under the followingconditions:

-   -   System: SMART System (Amersham Pharmacia Biotech)    -   Column: Mono Q PC 1.6/5 (0.16 cmφ×5 cm, 0.1 ml, Amersham        Pharmacia Biotech)    -   Buffer A: 50 mM Tris-HCl (pH 8.0)    -   Buffer B: 50 mM Tris-HCl (pH 8.0)/0.5 M NaCl

Sample was prepared by adding 0.1 ml of 1 M Tris base to the Protein Aelution fraction obtained by affinity chromatography to adjust the pH to8 to 9. The sample was loaded onto the column at a flow rate of 200μl/min, and then eluted by gradient elution using a gradient program of0% B/5 min, 0 to 60% B/30 min, 60 to 100% B/10 min and 100% B/10 min,with a flow rate of 50 μl/min. The eluate was collected as 50 μlfractions, and 2 to 4 fractions containing the antibody were combinedand subjected for activity measurement.

The affinity chromatography and anion exchange chromatography resultedin 0.5 to 1.0 μg of antibody. The anion exchange chromatogram of eachmutant is shown in FIGS. 3 to 5 and the superposed chromatograms of themutants are shown in FIG. 6. In addition, the amount and recovery rateof the proteins are shown in Table 2. The N54D mutant and N51D/N54Ddouble mutant were obtained as almost a single peak. However, subpeakwas observed for the original anti-human TF antibody, N51D mutant andN28D mutant. Particularly, N51D mutant showed 2 subpeaks, and that withhigh contents. TABLE 2 Initial Total protein protein Peak amountConcentration Dosage amount Recovery No. (μg) (ng/ml) (ml) (μg) (%)Original 1 4.9 6969.568 0.10 0.70 16.5 2 734.883 0.15 0.11 N28D 1 4.25436.713 0.15 0.82 20.7 2 320.086 0.15 0.05 N51D 1 3.8 2643.388 0.150.40 18.2 2 2724.396 0.10 0.27 3 143.479 0.15 0.02 N54D 3.9 2811.0460.20 0.56 14.4 N51D/ 3.4 5255.977 0.20 1.05 30.9 N54D5. Measurement of TF Binding Activity

TF binding activity was measured by competitive ELISA using biotinylatedanti-human TF antibody. Each of the anti-human TF mutant antibodies wasexpressed in COS-7, and purified using protein A affinity chromatographyand anion exchange chromatography to be used as samples. The subpeaksobserved during anion exchange chromatography of the original anti-humanTF antibody, N28D mutant and N51D mutant were used for the measurement.Lot No. 00C01 was used as the anti-human TF antibody standard.

shTF was adjusted to 20 nM with coating buffer (hereafter, indicated asCB), dispensed at 100 μl/well into a 96-well plate and incubated at 4°C. overnight. Each well was washed three times with rinse buffer(hereafter, indicated as RB), 200 μl dilution buffer (hereafter,indicated as DB) was added to each well. The plate was left standing atroom temperature for 2 hours for blocking. After discarding DB, 100 μlsample diluted by 2-fold serial dilution with DB containing 10,000-folddiluted biotinylated anti-human TF antibody was added to each well, andthe plate was left standing at room temperature for one hour. The platewas washed three times with RB, 100 μl ALP-streptavidin diluted5,000-fold with DB was dispensed to each well, and left standing for 1hour at room temperature. Each well was washed 5 times with RB andSIGMA104 adjusted with substrate buffer (hereafter, indicated as SB) to1 mg/ml was dispensed to each well. Plates were left standing for 30 minat room temperature for color development and measured with a microplatereader at a wavelength of 405 nm and a control wavelength of 655 nm.

The assessment of binding activity was performed as follows: astraight-line as the standard was obtained by linear-regression of theconcentration (logarithmic conversion value)-absorbance of the originalanti-TF human antibody. The absorbance of each sample within the rangeof 62.5 to 500 ng/ml was converted to standard antibody concentration(Cc) using this standard straight-line. The added antibody concentrationwas subtracted from Cc (Ca) to obtain the sample concentration ratio tothe standard antibody that shows the same binding activity as thebinding activity.

The measurement results on the binding activities are shown in FIG. 7and Table 3. The binding activity of each mutant was lower than that ofthe original anti-human TF antibody. The binding activity of the mutant(N54D mutant) of Asn54 located in CDR2 yet mostly expected to undergodeamidation decreased to about 10% of the original anti-human TFantibody. The binding activity of the mutant (N54D mutant) of Asn51located in CDR2 similar to Asn54 was about 50% of the originalanti-human TF antibody, and the degree of decrease was smaller than theN54D mutant. The N51D/N54D double mutant, a mutant of both the aminoacids Asn51 and Asn 54, had a further decreased binding activity thanthe N54D mutant. On the other hand, the binding activity of the mutant(N28D mutant) of Asn28 located in FR1 was about 94% of the originalanti-human TF antibody showing only a slight decrease. From thesefindings, deamidation of Asn51 and Asn54 located in CDR2, particularlyAsn54 was indicated to greatly reduce the binding activity.

Furthermore, the comparison of the binding activity of the subpeaks(peak 2) observed in the original anti-human TF antibody, N28D mutantand N51D mutant to that of the main peak (peak 1) revealed lower bindingactivity in all the subpeaks to the mainpeak. TABLE 3 Binding ActivityPeak 1 Peak 2 Native  100% 70.6% N28D 93.9% 46.3% N51D 49.2% 29.0% N54D 9.2% N51D/N54D  7.0%6. Measurement of TF Neutralizing Activity

TF neutralizing activity was measured using hTF (Thromborel S), FactorVIIa and Factor X. Similar to the measurement of the binding activity,each of the anti-human TF mutant antibodies was expressed in COS-7 cellsand purified using Protein A affinity chromatography and anion exchangecolumn chromatography. Lot No. 00C01 was used for the anti-human TFantibody standard.

Coagulation factor VIIa and Thromborel S were diluted with assay buffer(TBS (pH 7.49) containing 5 mM CaCl₂ and 0.1% BSA; hereafter, indicatedas AB) to 0.1 PEU/ml and 120-fold (v/v), respectively. 60 μl of thesemixtures were dispensed to each well of a plate and left standing for 60min at room temperature. ABX wherein Factor X is diluted with AB to 0.25PEU/ml was used to dilute the samples and 40 μl of sample diluted to thedesired concentration was dispensed to each well of the plate. The platewas left standing for 30 min at room temperature, and the reaction wasquenched by adding 10 μl/well of 500 mM EDTA. S-2222 mixture wasprepared by mixing one volume of S-2222, a chromogenic substrate,solution with one volume of MilliQ H₂O and two volumes of 0.6 mg/mlhexamethylene bromide solution. Fifty μl/well of the S-2222 mixture wasdispensed into the plate and left standing at room temperature. After 30min, measurements were performed using a micro plate reader at ameasurement wavelength of 405 nm and a control wavelength of 655 nm.

Measurement results on the neutralizing activity are shown in FIG. 8 andTable 4. The concentration of each of the mutant was calculated usingthe anti-human TF antibody standard as a standard, and the neutralizingactivity ratio compared to the anti-human TF antibody standard wasobtained. The neutralizing activity ratio bases on the concentration of250 ng/ml, at which concentration all samples could be measured. Theoriginal anti-human TF antibody and N28D mutant retained a neutralizingactivity almost equivalent to the anti-human TF antibody standard. Thus,the deamidation of Asn28 located in FR was considered not to affect thedecrease of neutralizing activity.

On the other hand, the neutralizing activity ratios of N51D and N54Dmutants against the anti-human TF antibody standard decreased to 65.6%and 19.9%, respectively. Therefore, the deamidation of Asn51 and Asn54located in CDR of the anti-human TF antibody was strongly suggested tocause the decrease of neutralizing activity. TABLE 4 Added CalculatedNeutralizing concentration concentration activity ratio (ng/ml) (ng/ml)(%) N28D 250 253 101 N51D 250 164 65.6 N54D 250 49.8 19.9 N51D/N54D 25031.3 12.5 Native 250 248 99.1

From the results described above, the solution pH-dependent decrease inthe binding activity to TF and increase in low pI molecular species ofunformulated anti-human TF antibody under antibody destabilizingconditions were revealed to mainly result from the deamidation of Asn54in the CDR2 region.

EXAMPLE 2 Measurement of TF Binding and Neutralizing Activities of Gly55Substitution Mutant of Anti-Human TF Antibody

The anti-human TF antibody described in WO 99/51743 contains thehumanized heavy chain version i (SEQ ID NO: 25, FIG. 1) and thehumanized light chain version b2 (SEQ ID NO: 26, FIG. 1). Based on itsamino acid sequence, mutants were prepared wherein the Gly55 in theheavy chain CDR2 that is considered as an important amino acid in theconstruction of the loop of CDR2 had been substituted with 19 otheramino acids. Then, the binding activity of each mutant with TF wasmeasured. Furthermore, the neutralizing activity and deamidation wasobserved for the mutants wherein Gly55 had been substituted with Ile,Leu, Phe, Glu and Lys.

The amino acid sequence of the antibody based on the sequence describedby Kabat et al. (Kabat E. A., Wu T. T., Perry H. M., Gottesman K. S. andFoeller C., “Sequences of proteins of immunological interest. 5th ed.”,US Dept. Health and Human Services, Bethesda, Md., 1991).

1. Construction of Anti-Human TF Mutant Antibody Expression Vector

The cloning vector pCVIDEC-AHi (FIG. 9A) and the anti-human TF antibodyexpression vector pN5KG4P-AHi-ALb2 carrying the heavy chain variableregion (AHi) of the anti-human TF antibody were isolated from E. coliSCS110 (dam⁻/dcm⁻¹).

The substitution of the codon encoding Gly55 with a different amino acidwas performed on the pCVIDEC-AHi. In this procedure, the substitution to15 amino acids wherein the third codon can be fixed to “C” was performedas follows: digesting a fragment of about 30 bp comprising the codingregion of Asn54-Gly55 at the unique sites XbaI and BalI of pCVIDEC-AHi,and integrating a fragment prepared using a synthetic oligo DNA whereinthe 2 nucleotides at the 3′-end of the Gly55-coding codon has beenrandomized (FIG. 9B). The XbaI-BalI fragment was prepared by elongatingthe 3′-end with two nucleotides using Vent polymerase (NEB, Inc.) sothat the 1st and 2nd nucleotides of the Gly55 codon in the CDR2 variableof the of the anti-human TF antibody heavy chain region become randomnucleotide sequences, and then digesting with XbaI. This procedure wasbelieved to enable production of 15 mutants with high codon usage inmammals via one operation. However, in fact, only 8 kinds of mutantswere produced since optimal reaction conditions could not be found.Therefore, the remaining mutants were constructed using otherrestriction enzyme sites.

Mutants comprising substitution of an amino acid wherein the 3^(rd)codon of Gly55 has to be converted, as well as those that could not beproduced by the above-descried method were produced as follows: a vectorwherein the EcoRI site of pCVIDEC-AHi is changed to HindIII site wasconstructed, digested at the unique sites ApoI and XbaI of pCVIDEC-AHi,and a fragment produced using synthetic oligo DNA was inserted. That is,apart from the XbaI and BalI sites, ApoI and XbaI sites were thepossible sites that can be used as the restriction enzyme sites.However, ApoI also digests the ECORI sites in the vector. Therefore, theEcoRI site was first removed by changing it to a HindIII site. TheApoI-XbaI fragment was about 55 bp. Thus, a synthetic oligo DNA wasprepared so that a total of about 16 bp overlap upstream and downstreamof the nucleotide sequence of the codon encoding Gly 55 that is changedto other amino acids. After annealing, the fragment was elongated usingVent polymerase and digested with ApoI and XbaI (FIG. 9C and Table 5).TABLE 5 EcoRI site deletion adapter                 Hind III       G  AATTC    AATTGGAAGCTTGC        CTTAA  G        CCTTCGAACGTTAAH-G56M primer-F GAGTCTAGAATGGATTGGTGGGAATGATCCTGCGAATATGC H-G56Mprimer-R GAGAATTTCGGGTCATACATACTATGCATATTCGCAGGAT H-G56M primer-FGAGTCTAGAATGGATTGGTGGGAATGATCCTGCGAATAAGCAT H-G56K primer-RGAGAATTTCGGGTCATACATACTATGCTTATTCGCAGGAT H-G56W primer-FGAGTCTAGAATGGATTGGTGGGAATGATCCTGCGAATTGGCAT H-G56W primer-RGAGAATTTCGGGTCATACATACTATGCCAATTCGCAGGAT H-G56Q primer-FGAGTCTAGAATGGATTGGTGGGAATGATCCTGCGAATCAGCAT H-G56Q primer-RGAGAATTTCGGGTCATACATACTATGCTGATTCGCAGGAT H-G56E primer-FGAGTCTAGAATGGATTGGTGGGAATGATCCTGCGAATGAGCAT H-G56E primer-RGAGAATTTCGGGTCATACATACTATGCTCATTCGCAGGAT H-G56F primer-FGAGTCTAGAATGGATTGGTGGGAATGATCCTGCGAATTTCCAT H-G56F primer-RGAGAATTTCGGGTCATACATACTATGGAAATTCGCAGGAT H-G56T primer-FGAGTCTAGAATGGATTGGTGGGAATGATCCTGCGAATACCCAT H-G56T primer-RGAGAATTTCGGGTCATACATACTATGGGTATTCGCAGGAT H-G56N primer-FGAGTCTAGAATGGATTGGTGGGAATGATCCTGCGAATAACCAT H-G56N primer-RGAGAATTTCGGGTCATACATACTATGGTTATTCGCAGGAT H-G56D primer-FGAGTCTAGAATGGATTGGTGGGAATGATCCTGCGAAGACCAT H-G56D primer-RGAGAATTTCGGGTCATACATACTATGGTCATTCGCAGGAT H-G56P primer-FGAGTCTAGAATGGATTGGTGGGAATGATCCTGCGAATCCCCAT H-G56P primer-RGAGAATTTCGGGTCATACATACTATGGGGATTCGCAGGAT H-G56C primer-FGAGTCTAGAATGGATTGGTGGGAATGATCCTGCGAATTGCCAT H-G56C primer-RGAGAATTTCGGGTCATACATACTATGGCAATTCGCAGGATPrimers for the construction of anti-human TF mutant antibodies usingXbaI and ApoI sites are shown.

In addition, the nucleotide sequences indicated in Table 5 are shown inSEQ ID NOs: 1 to 24.

The sequences of the constructed 19 different anti-human TF mutantantibodies were confirmed in the cloning vector by a sequencer.Furthermore, the sequences were reconfirmed after constructing mutantexpression vectors by replacing the heavy chain variable region obtainedthrough NheI and SalI digestion with the heavy chain variable regiondigested from the anti-human TF antibody expression vector with NheI andSalI. After confirming that the target sequence was obtained, theanti-human TF mutant antibody expression vector was amplified using E.coli DH5α, purified using QIAGEN Maxi column and the sequence wasconfirmed. As a result, 19 different anti-human TF mutant antibodyexpression vectors were obtained.

2. Transient Expression of Anti-Human TF Mutant Antibody in CHO Cells

A total of 20 expression vectors, i.e., the constructed anti-humanantibody heavy chain Gly55 mutant expression vectors and Gly55non-substituted antibody (Gly55Gly) expression vector, were transfectedinto CHO cells via the lipofection method for transient expression. Aday before lipofection, the CHO (dhfr-) cells were cultured on 10%FCS-α-MEM in an atmosphere of 5% CO₂ at 37° C. The CHO cells were seededat 1×10⁵ cells/well on 12-well plates and cultured at 5% CO₂ at 37° C.

After adding 6 μl of FuGENE6 Transfection Reagent (Boehringer MannheimGmbH) to 100 μl of Opti-MEM (GIBCO BRL) and leaving standing for 5 min,the mixture was added to a tube containing 1 μg of each of theanti-human TF antibody heavy chain Gly55 mutant expression vectorspN5KG4P-AHi-Alb2-G55X (X: 20 kinds of each amino acid). The tube wasleft standing for 20 min to form a FuGENE6/DNA complex. After discardingthe media of the CHO cell seeded on the previous day, 2 ml/well of 10%FCS-α-MEM was newly added followed by the addition of each FuGENE6/DNAcomplex in triplicate.

The cells were cultured at 37° C. at 5% CO₂ for one day and then washedwith PBS. Media were replaced by adding 3 ml/well of 10% FCS-α-MEM.After 7-day incubation at 5% CO₂ at 37° C., about 9 ml of culturesupernatant containing each of the anti-human TF mutant antibody wastransferred into a 15 ml tube, centrifuged at 1000 rpm for 5 min, andconcentrated to 10-fold through ultrafiltration. The obtained culturesupernatant was used as the anti-human TF antibody heavy chain Gly55Xaamutant sample.

3. Measurement of TF Binding Activity

Human IgG content in the anti-human TF antibody heavy chain Gly55Xaamutant sample of was measured to adjust the IgG concentration of eachsample to 100 ng/ml.

TF binding activity was measured by competitive ELISA using biotinylatedanti-human TF antibody. shTF was adjusted to 20 nM with CB, dispensed at100 μl/well into a 96-well plate, and was left standing at 4° C.overnight. The plate was washed three times with RB, 200 μl/well of DBwas dispensed thereto, and then left standing for blocking at roomtemperature for 2 hours. After removing DB, 100 μl/well of standard andsamples diluted by 2-fold serial dilution with DB containingbiotinylated anti-human TF antibody (diluted 10,000-fold at finalconcentration) were added. The plates were left standing at roomtemperature for 0.1 hour. After washing 3 times with RB, 100 μl/well ofALP-streptavidin, diluted 8,000-fold with DB, was added and leftstanding at room temperature for one hour. SIGMA104 adjusted to 1 mg/mlwith SB was added after washing 3 times with RB, and left standing forabout 20 min at room temperature to develop colors and measure with amicroplate reader at a wavelength of 405 nm and a control wavelength of655 nm.

The binding activity was compared by determining the concentration thatshowed 50% activity according to the following procedure: the absorbanceat each measured point was converted to percentage (%) by taking theabsorbance of sample (−) and biotinylated antibody (+) as 100%. A linearregression equation of “concentration (logarithmic conversionvalue)−absorbance (%)” was obtained based on two points which sandwichthe 50% value of each sample. Then, the concentration giving 50%absorbance was calculated to calculate the binding activity of eachsample from Equation 1.binding activity=(50% activity concentration of the standardantibody)/(50% activity concentration of sample)×100.  Equation 1

The anti-human TF antibody concentration conversion value that wascalculated based on the calibration curve produced using bulk anti-humanTF antibody (Lot No. 00C01) is shown in FIG. 10. The Gly55non-substituted antibody (Gly55Gly) expressed in CHO cells retained a TFbinding activity almost equivalent to the bulk anti-human TF antibody.Decrease in the binding activity was observed in the Gly55 mutants,Gly55Val, Gly55Ile and Gly55Pro.

The following assay was performed to examine the TF binding activity ofthe anti-human TF antibody heavy chain Gly55 mutants in detail.Specifically, the TF binding activity was measured by competitive ELISAmethod using the anti-human TF mutant antibodies by changing the amountof added sample within the range of 25 to 200 ng/ml. Measurements onGly55Asn and Gly55Asp were not performed due to the lack of sampleamount.

The measurement results are shown in FIG. 11. Among the examined 18anti-human TF antibody heavy chain Gly55 mutants, the TF bindingactivity of Gly55Val, Gly55Ile and Gly55Pro were significantly decreasedcompared with the bulk anti-human TF antibody (Lot No. 00C01) and Gly55non-substituted antibody (Gly55Gly). However, no significant differencein the TF binding activity could be observed for the other 15 mutants.Thus, the TF binding activity was supposed to be maintained even afterchanging the Gly55 with another amino acid.

4. Measurement of TF Neutralizing Activity

Coagulation factor VIIa and Thromborel S were diluted with AB to 0.1PEU/ml and 120-fold (v/v), respectively. Sixty μl/well of a mixturethereof was dispensed to a plate and left standing at room temperaturefor 60 min. Twenty μl/well of sample diluted by 2-fold serial dilutionwith 10 mM phosphate buffer was dispensed followed by 20 μl/wellcoagulation factor (Factor X) solution diluted to 0.5 PEU/ml with AB(supplemented with CaCl₂ solution to a CaCl₂ concentration of 10 mM).The plate was left standing at room temperature for 30 min and then thereaction was quenched by adding 10 μl/well of 500 mM EDTA. Fifty μl/wellof a solution of test-team chromogenic substrate S-2222 solution andpolybrene solution mixed at 1:1 was dispensed and left standing at roomtemperature. After 30 min, measurements were taken by a microplatereader at a measurement wavelength of 405 nm and a control wavelength of655 nm.

The neutralizing activity was compared by determining the concentrationshowing 50% activity according to the following procedure: theabsorbance at each measured point was converted to percentage (%) bytaking the absorbance of sample (−) and coagulation factor X (+) as100%, and sample (−) and coagulation factor X (−) as 0%. A linearregression equation of “concentration (logarithmic conversionvalue)−absorbance (%)” was obtained based on two points that sandwichthe 50% value of each sample. Then, the concentration giving 50%absorbance was calculated to calculate the neutralizing activity of eachsample from Equation 2.Neutralizing activity (IC50)=(50% activity concentration of the standardantibody)/(50% activity concentration of sample)×100.  Equation 25. Construction of Stable Expression System of Anti-Human TF MutantAntibodies Using CHO Cells

Five kinds of mutants, Gly55Leu, Gly55Phe, Gly55Glu, Gly55Lys andGly55Ile, wherein the Gly55 is substituted to Leu (aliphatic aminoacid), Phe (aromatic amino acid), Glu (acidic amino acid), Lys (basicamino acid) and Ile (branched-chain aliphatic amino acid), respectively,were produced in sufficient quantity to compare the activity of theanti-human TF mutant antibodies by constructing stable expression celllines.

5-1 Gene Transfer into CHO Cells

CHO (dhfr-) cells were washed with PBS and then resuspended in PBS toabout 1×10⁷ cells/ml. The cells were transferred into a 0.4 cm cuvettetogether with 10 μg of the expression vector of the anti-human TFantibody heavy chain Gly55 mutant, pN5KG4P-AHi-Alb2-G55X.Electroporation was performed at 1.5 kV with 25 μF. After leavingstanding for 10 min, the cells were suspended in 200 ml of 10% FCS-α-MEMnucleic acid (−) media. Two hundred μl/well of the suspension was seededand cultured on ten 96-well plates.

5-2 Selection of Transfected Cells

The amount of expressed antibody in wells wherein cell growth could beobserved during the 96-well plate culture was compared by hIgG ELISA.Cells that showed high hIgG expression were subcultured from 10 wellseach at a total of 70 wells into 12 well plates and cultured in 10%FCS-α-MEM nucleic acid (−) media. The expression amount of anti-human TFmutant antibody was measured by hIgG ELISA at the time when the cellshad acclimatized to the 10% FCS-α-MEM nucleic acid (−) media and showedsatisfactory growth. Four wells were selected for each mutant andsubcultured into a 50 ml flask. Antibody production was enhanced byreplacing the media with 10% FCS-α-MEM nucleic acid (−) containing 10 nMMTX.

5-3 Production of Anti-Human TF Mutant Antibody by Large-Scale CultureUsing Serum Free Media

Among the anti-human TF mutant antibody clones, one clone each for eachmutant having a high hIgG expression level was selected and cultured insix 175 cm² flasks using media containing 10 nM MTX. The media werereplaced with 150 ml CHO-S-SFM II serum free media after reachingsubconfluence and incubated for 7 days. The culture supernatant wascollected, treated with 0.22 μm filter, and stored at −80° C. untilpurification.

5-4 Measurement of Expression Level of Anti-Human TF Mutant Antibody bySandwich ELISA

One hundred μl/well of anti-human IgG (γ) antibody was dispensed into a96-well plate and left standing at 4° C. overnight. After washing 3times with RB, 200 μl/well of DB was dispensed and left standing at roomtemperature for 2 hours for blocking.

After discarding DB, 100 μl/well of the standard and sample that wasproperly diluted with DB or media used to recover the antibody from theanti-human TF mutant antibody producing cells was added, and leftstanding at room temperature for 2 hours. After washing three times withRB, 100 μl/well of HRP-labeled anti-human IgG antibody diluted10,000-fold with DB was dispensed and left standing at room temperaturefor 1 hour. After washing 10 times with RB, 100 μl/well of chromogenicreagent was dispensed and left standing at room temperature for about 10min. Color reaction was quenched by the addition of 50 μl/well of 2 Nsulfuric acid to measure the absorbance with a microplate reader at ameasurement wavelength of 450 nm and a control wavelength of 655 nm.

Consequently, several milligrams of each of the anti-human TF mutantantibodies, except Gly55Gly, were obtained (Table 6). TABLE 6 VersionG55G G55F G55L G55E G55K G55I 99D01 Clone No. 196 41 96 23 237 127 α-MEMN (−) (ng/ml) 29 64 9 59 600 110 α-MEM 10 nM 50 836 3451 6143 423 369MTX (ng/ml) CHO-SFM-II (large-Scale: 0.24 15.4 41.7 50 11.5 5.8 900 ml)(μg/ml) Purified — 319 379 624 180 153 1556 (after buffer exchange: (4ml) μg/ml, total 7 ml)6. Purification of Each Anti-Human TF Antibody Mutant

Each mutant was purified from the supernatant of the large scale culturecontaining each of the mutants using a HiTrap rProtein A FF column and aHiTrap Q Sepharose HP column.

6-1 Affinity Chromatography for Purification

Affinity chromatography was performed in a refrigerated room under thefollowing conditions:

-   System: FPLC System-   Column: HiTrap rProtein A FF (1.6 cmφ×2.5 cm, 5 ml)-   Equilibrating buffer: D-PBS (−)-   Washing buffer: 10 mM Sodium phosphate buffer (pH 7.4)-   Elution buffer: 50 mM Acetic acid (pH 2 to 3)

Samples were loaded onto the column after adjusting the pH to 7.4 with0.5 M disodium phosphate solution. Fifty ml of washing buffer was usedto dilute 1.5 ml (16.5 mg) of the anti-human TF antibody standard.Elution was performed with 25 ml (5 C.V.) elution buffer at a flow rateof 5 ml/min and the pH was neutralized to 6 to 7 with 1.25 ml of 1 MTris base.

6-2 Anion Exchange Chromatography for Purification

Next, anion exchange chromatography was conducted in a refrigerated roomunder the following conditions:

-   System: FPLC System-   Column: HiTrap Q Sepharose HP (0.7 cmφ×2.5 cm, 1 ml)-   Buffer A: 50 mM Tris-HCl (pH 8.0, 4° C.)-   Buffer B: 50 mM Tris-HCl (pH 8.0, 4° C.)/1 M NaCl

Sample was prepared by adjusting the pH of the Protein A elutionfraction obtained via affinity chromatography to 8 to 9 through theaddition of 1.25 ml of 1 M Tris base. Elution steps of 0 mm NaCl (5C.V.), 250 mM NaCl (5+5 C.V.) and 1 M NaCl (100 C.V.) at a flow rate of1 ml/min were performed and the first half 5 C.V. (5 ml) of the 250 mMNaCl step was collected.

Five hundred μg or more of each of the anti-human TF mutant antibodies,except Gly55Gly, was obtained (Table 5). Gly55Gly was not obtained.Therefore, bulk anti-human TF antibody (Lot No. 99D01) was purifiedaccording to a similar procedure to use it for comparison withanti-human TF antibody (Table 5).

7. Anion Exchange Chromatography for Analysis

Sample was analyzed by anion exchange chromatography at room temperatureunder the following conditions:

-   -   System: SMART System    -   Column: MonoQ PC 1.6/5 (0.16 cmφ×5 cm, 0.1 ml)    -   Buffer A: 50 mM Tris-HCl (pH 8.0, 20° C.)    -   Buffer B: 50 mM Tris-HCl (pH 8.0, 20° C.)/500 mM NaCl

Gradient elution with a gradient program of 0% B/5 min, 0-60% B/30 min,60 to 100% B/10 min and 100% B/10 min at a flow rate of 50 μl/min wasperformed. Two μg of sample (calculated by UV conversion) was diluted 3to 50 times with 50 μl of buffer A and 25 μl thereof was analyzed.

The analysis by anion exchange chromatography of purified bulkanti-human TF antibody (99D01) and each of the anti-human TF mutantantibody revealed almost a single peak, although with a change inelution time depending on the introduced amino acid mutation (FIG. 12).

8. Suppression of Anti-Human TF Antibody Deamidation by Amino AcidMutation

In order to examine the deamidation reaction, accelerated testing wasperformed under heated condition using a neutral pH buffer whereindeamidation easily occurs.

8-1 Replacement of Buffer

Replacement of sample buffer with 20 mM sodium phosphate buffer/150 mMsodium chloride (pH 7.5) buffer using a PD-10 desalting column wasperformed. After equilibrating the column, 2.5 ml sample was loaded ontotwo columns and eluted with 3.5 ml buffer.

8-2 Sample Preparation for Accelerated Testing

Each sample of the anti-human TF mutant antibodies was diluted to 100μg/ml based on the value quantitated by hIgG ELISA. Buffer containing 20mM sodium phosphate buffer/150 mM NaCl (pH 7.5) was used. After passingthrough a 0.22 μm filter, 1 ml of each sample was dispensed into a 5 mlvial.

8-3 Accelerated Testing

Accelerated testing on the purified bulk anti-human TF antibody (99D01)and anti-human TF mutant antibodies was performed for four weeks at 40°C. in 20 mM sodium phosphate buffer/150 mM NaCl (pH 7.5) solution. Aportion was sampled at each point of 0, 1, 2 and 4 weeks, and itsactivity was analyzed through the comparison of TF binding activity andTF neutralizing activity. Deamidation at each point was analyzed usinganalytical anion exchange chromatography.

The value (Table 7) obtained by requantitation using the monomerfraction of GPC as an indicator was used for the comparison of activity.Specifically, quantitation of antibody was performed at room temperatureunder following conditions:

-   -   System: Waters (600S Controller, 616 Pump, 486 Tunable        absorbance detector, 717 Plus Autosampler)    -   Column: TSK gel G3000SWXL (0.78 cmφ×30 cm, guard column 0.6        cmφ×4 cm)    -   Buffer: 50 mM Sodium phosphate/300 mM NaCl (pH 7.0)

Analysis was performed at a flow rate of 0.5 ml/min using 100 μl(equivalent to 10 μg) of accelerated material as a sample. TABLE 7Initial value 1 week 2 weeks 4 weeks 99D01 116.1 115.2 116.5 112.4 G55L116.5 113.8 115.6 113.7 G55I 102.7 99.6 98.4 94.5 G55F 118.3 115.3 114.8111.8 G55E 110.8 110.2 110.7 109.8 G55K 135.2 134.9 136.0 130.9

Similar to transiently expressed anti-human TF mutant antibodies, theresult showed that the TF binding activity of Gly55Ile before theaccelerated testing was about 26% of the bulk anti-human TF antibody(Lot No. 00C01). Namely, the activity was low and significantly reducedcompared with 99D01 (FIG. 13A). Almost an equivalent activity to 99D01was retained by Gly55Leu, Gly55Glu, Gly55Phe and Gly55Lys (FIG. 13A).After 4 weeks of accelerated testing, the anti-human TF mutantantibodies retained 80% or more of its activity before the acceleratedtesting, whereas the activity of 99D01 decreased to about 60% of itsactivity before the accelerated testing (FIG. 13B).

The analysis of deamidation using analytical anion exchangechromatography indicated significant increase in a peak considered tocorrespond to the deamidated molecule in 99D01 but almost none in theanti-human TF mutant antibodies (FIG. 14). In the interest of thechanges in the TF neutralizing activity over time, 99D01 showed arelatively large reduction in activity (FIG. 15).

From these results, deamidation of Asn55 was determined to be suppressedby the substitution of Gly55, and amino acid substitution Gly55Leu andGly55Phe were suggested suitable for suppressing deamidation.

9. TF Neutralizing Activity Before and After Accelerated Testing

TF neutralizing activities of 99D01 and each of the anti-human TF mutantantibodies are shown in FIG. 16. Although Gly55Glu and Gly55Ile showedlow activities of about 41% and about 13%, respectively, the other 3anti-human TF mutant antibodies had activities between 56 to 74%, i.e.,nearly the same as 99D01 (66%).

The IC50 value compared to the initial value of each sample wascalculated from FIG. 15 in order to examine the amount of activitydecrease over time of 99D01 and each of the anti-human TF mutantantibodies in the accelerated testing (FIG. 17). Since the IC50 valueafter the accelerated testing at 40° C. for 4 weeks could not becalculated for the Gly55Ile sample, the results up to 2 weeks areindicated for this sample. The TF neutralizing activity of 99D01 afterthe accelerated testing at 40° C. for 4 weeks decreased to about 40% ofthe initial value. On the other hand, each of the mutants wherein Gly55was substituted with another amino acid maintained a TF neutralizingactivity of 50 to 70% of the initial value even after the acceleratedtesting at 40° C. for 4 weeks.

These results indicate that substitution of a glycine that is locatedadjacent to an asparagine in an antibody with another amino acid doesnot decrease the antibody activity, yet it suppresses instability due todeamidation.

Industrial Applicability

The present inventors found that substitution of glycine that is locatedadjacent to asparagine with another amino acid does not influence theantibody activity. The present invention can be applied to produceantibodies showing low activity decrease, and thus, to obtain antibodiesthat can be used as pharmaceutical agents that are required to be stablefor a long time. Furthermore, the present invention can also be appliedto proteins other than antibodies, and are expected to achievesuppression of deamidation without affecting the protein activity.

1. A method for stabilizing a protein, which comprises the step ofsubstituting an amino acid that is located adjacent to an amino acidbeing deamidated with another amino acid.
 2. The method for stabilizinga protein of claim 1, wherein the amino acid being deamidated isasparagine.
 3. The method for stabilizing a protein of claim 1, whereinthe amino acid that is located adjacent to the C-terminal side of theamino acid being deamidated is glycine.
 4. The method for stabilizing aprotein of any one of claims 1 to 3, wherein the protein is an antibody.5. The method for stabilizing a protein of claim 4, wherein the antibodyis humanized antibody.
 6. The method for stabilizing a protein of claim4 or 5, wherein the amino acid being deamidated exists in thecomplementary determining region (CDR).
 7. The method for stabilizing aprotein of claim 6, wherein the complementary determining region (CDR)is CDR2.
 8. The method for stabilizing a protein of any one of claims 1to 3, wherein the protein is an antigen binding protein.
 9. The methodfor stabilizing a protein of any one of claims 1 to 3, wherein theprotein belongs to the immunoglobulin superfamily.
 10. The method forstabilizing a protein of any one of claims 1 to 3, wherein the proteinis a pharmaceutical agent.
 11. A protein stabilized by the method of anyone of claims 1 to
 10. 12. The stabilized protein of claim 11 whoseantigen binding activity is 70% or more of the activity before the aminoacid substitution.